Surface enhanced spectroscopy-active composite nanoparticles

ABSTRACT

Metal nanoparticles associated with a spectroscopy-active (e.g., Raman-active) analyte and surrounded by an encapsulant are useful as sensitive optical tags detectable by surface-enhanced spectroscopy (e.g., surface-enhanced Raman spectroscopy).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/345,821, filed Jan. 16, 2003, entitled entitled “Surface EnhancedSpectroscopy-Active Composite Nanoparticles,” which is acontinuation-in-part of U.S. patent application Ser. No. 09/680,782,filed Oct. 6, 2000, entitled “Surface Enhanced Spectroscopy-ActiveComposite Nanoparticles,” now U.S. Pat. No. 6,514,767, issued Feb. 4,2003, incorporated herein by reference, which claims priority to U.S.Provisional Application No. 60/157,931, filed Oct. 6, 1999, entitled“Glass Coated Surface Enhanced Raman Scattering Tags,” and U.S.Provisional Application No. 60/190,395, filed Mar. 17, 2000, entitled“GANS Particles.”

FIELD OF THE INVENTION

This invention relates generally to submicron-sized tags or labels thatcan be covalently or non-covalently affixed to entities of interest forthe purpose of quantification, location, identification, or tracking.More particularly, it relates to surface enhanced spectroscopy-activecomposite nanoparticles, methods of manufacture of the particles, anduses of the particles.

BACKGROUND OF THE INVENTION

When light is directed onto a molecule, the vast majority of theincident photons are elastically scattered without a change infrequency. This is termed Rayleigh scattering. However, the energy ofsome of the incident photons (approximately 1 in every 10⁷ incidentphotons) is coupled into distinct vibrational modes of the molecule'sbonds. Such coupling causes some of the incident light to beinelastically scattered by the molecule with a range of frequencies thatdiffer from the range of the incident light. This is termed the Ramaneffect. By plotting the frequency of such inelastically scattered lightagainst its intensity, the unique Raman spectrum of the molecule underobservation is obtained. Analysis of the Raman spectrum of an unknownsample can yield information about the sample's molecular composition.

The incident illumination for Raman spectroscopy, usually provided by alaser, can be concentrated to a small spot if the spectroscope is builtwith the configuration of a microscope. Since the Raman signal scaleslinearly with laser power, light intensity at the sample can be veryhigh in order to optimize sensitivity of the instrument. Moreover,because the Raman response of a molecule occurs essentiallyinstantaneously (without any long-lived highly energetic intermediatestates), photobleaching of the Raman-active molecule—even by this highintensity light—is impossible. This places Raman spectroscopy in starkcontrast to fluorescence spectroscopy, in which photobleachingdramatically limits many applications.

The Raman effect can be significantly enhanced by bringing theRaman-active molecule(s) close (≦50 Å) to a structured metal surface;this field decays exponentially away from the surface. Bringingmolecules in close proximity to metal surfaces is typically achievedthrough adsorption of the Raman-active molecule onto suitably roughenedgold, silver or copper or other free electron metals. Surfaceenhancement of the Raman activity is observed with metal colloidalparticles, metal films on dielectric substrates, and with metal particlearrays. The mechanism by which this surface-enhanced Raman scattering(SERS) occurs is not well understood, but is thought to result from acombination of (i) surface plasmon resonances in the metal that enhancethe local intensity of the light, and; (ii) formation and subsequenttransitions of charge-transfer complexes between the metal surface andthe Raman-active molecule.

SERS allows detection of molecules attached to the surface of a singlegold or silver nanoparticle. A Raman enhancing metal nanoparticle thathas associated or bound to it a Raman-active molecule(s) can haveutility as an optical tag. For example, the tag can be used inimmunoassays when conjugated to an antibody against a target molecule ofinterest. If the target of interest is immobilized on a solid support,then the interaction between a single target molecule and a singlenanoparticle-bound antibody can be detected by searching for theRaman-active molecule's unique Raman spectrum. Furthermore, because asingle Raman spectrum (from 100 to 3500 cm⁻¹) can detect many differentRaman-active molecules, SERS-active nanoparticles may be used inmultiplexed assay formats.

SERS-active nanoparticles with adsorbed Raman-active molecules offer thepotential for unprecedented sensitivity, stability, and multiplexingfunctionality when used as optical tags in chemical assays. However,metal nanoparticles present formidable practical problems when used insuch assays. They are exceedingly sensitive to aggregation in aqueoussolution; once aggregated, it is not possible to re-disperse them. Inaddition, the chemical compositions of some Raman-active molecules areincompatible with the chemistries used to attach other molecules (suchas proteins) to metal nanoparticles. This restricts the choices ofRaman-active molecules, attachment chemistries, and other molecules tobe attached to the metal nanoparticle.

SUMMARY OF THE INVENTION

One embodiment of the present invention provides surface-enhancedspectroscopy (SES)-active composite nanoparticles, such as SERS-activecomposite nanoparticles (SACNs). Such nanoparticles each contain aSES-active metal nanoparticle; a submonolayer, monolayer, or multilayerof spectroscopy-active species associated with or in close proximity(e.g., adsorbed) to the metal surface; and an encapsulating shell madeof a polymer, glass, or any other dielectric material. This places thespectroscopy-active molecule (alternately referred to herein as the“analyte,” not to be confused with the species in solution that isultimately being quantified) at the interface between the metalnanoparticle and the encapsulant.

In some embodiments, the encapsulant is glass. The resultingglass-coated analyte-loaded nanoparticles (GANs) retain the activity ofthe spectroscopy-active analyte, but tightly sequester this activityfrom the exterior surface of the nanoparticle. Thus, in the case ofsurface-enhanced Raman scattering (SERS), the resulting GAN exhibitsSERS activity, but the Raman-active analyte is located at the interfacebetween the metal nanoparticle and the encapsulant.

The analyte molecule can be chosen to exhibit extremely simple Ramanspectra, because there is no need for the species to absorb visiblelight. This, in turn, allows multiple composite nanoparticles, each withdifferent analyte molecules, to be fabricated such that the Ramanspectrum of each analyte can be distinguished in a mixture of differenttypes of particles.

Surface-enhanced spectroscopy (SES)-active composite nanoparticles areeasily handled and stored. They are also aggregation resistant,stabilized against decomposition of the analyte in solvent and air,chemically inert, and can be centrifuged and redispersed without loss ofSERS activity. SACNs may be provided as a dispersion in suitable solventfor storage or association with an object or molecule.

In one embodiment, the encapsulant may be readily derivatized bystandard techniques. This allows the particles to be conjugated tomolecules (including biomolecules such as proteins and nucleic acids) orto solid supports without interfering with the Raman activity of theparticles. Unlike metal nanoparticles, SACNs can be evaporated todryness and then completely redispersed in solvent. Using the techniquesprovided herein, it is possible to fabricate particles that areindividually detectable using SERS.

In an alternative embodiment, SACNs are attached to, mixed with, orotherwise associated with objects for tracking, identification, orauthentication purposes. Each type of particle or group of particletypes, as defined by the Raman spectrum of the encapsulated analyte,represents a particular piece of information. Tagged objects areverified by acquiring their Raman spectrum. Any liquid, solid, orgranular material can be tagged with a SACN. By associating an objectwith more than one different SACN type, a large number of distinct SACNgroups can be obtained.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a transmission electron micrograph of GANs with 35 nm Aucores and 40 nm glass shells. FIG. 1B is a transmission electronmicrograph of GANs with 35 nm Au cores and 16 nm glass shells.

FIG. 2 is a transmission electron micrograph of 35 nm Au, 8 nm glassGANs following centrifugation in a 50% glycerol solution.

FIGS. 3A and 3B are plots of absorbance versus wavelength, for differentetch times, and absorbance versus etch time at a single wavelength,respectively, for a particle having a 35 nm Au core and 8 nm glassshell.

FIG. 4 shows Raman spectra of GANs with a 40 nm Au core encapsulated in4 mm of glass, with (trace A) and without (trace B) the Raman-activeanalyte, trans-1,2-bis(4-pyridyl)ethylene (BPE).

FIG. 5 shows Raman spectra of a suspension of GANs with 40 nm Au coatedwith BPE and 4 nm glass encapsulant (Trace A); supernatant from a firstcentrifugation of the GANs (Trace B); and supernatant from a secondcentrifugation of the GANs (Trace C).

FIG. 6 shows Raman spectra of GANs (80 nm Au core/2-mercaptopyridine/20nm glass) and of a 50 mM solution of 2-mercaptopyridine absorbed onto aconventional three-layer SERS substrate.

FIG. 7 shows Raman spectra of the following four types (“flavors”) ofGANs particles: (A) GANs tagged with furonitrile; (B) GANs tagged withfuronitrile (66%) and cyanoacetic acid (33%); (C) GANs tagged withfuronitrile (33%) and cyanoacetic acid (66%); and (D) GANs tagged withcyanoacetic acid.

FIG. 8 shows Raman spectra of GANs (40 nm Au core/4 nm glass) (a) taggedwith BPE; (b) tagged with imidazole; or (c) untagged.

FIGS. 9A and 9B show Raman spectra of GANs containing BPE andpara-nitroso-N,N′-dimethylaniline (p-NDMA) in ethanol spotted onto whiteand yellow paper, respectively.

DETAILED DESCRIPTION OF EMBODIMENTS

Various embodiments of the present invention are directed tosurface-enhanced spectroscopy-active composite nanoparticles (SACNs),including surface-enhanced Raman spectroscopy (SERS)-active compositenanoparticles. Other embodiments provide methods of manufacture of theparticles and methods of use of the particles. These submicron-sizedtags or labels can be covalently or non-covalently affixed to or mixedwith entities of interest, ranging in size from molecular tomacroscopic, for the purpose of quantification, location,identification, or tracking.

The SACNs provided by embodiments of the present invention are uniquelyidentifiable nanoparticles. They can be used in virtually any situationin which it is necessary to label molecules or objects with an opticaltag. Biomolecules can be conjugated readily to the exterior of SACNs bystandard techniques, thereby allowing the particles to function asoptical tags in biological assays. SACNs can be used in virtually anyassay that uses an optical tag, such as a fluorescent label. However, asoptical tags, SACNs have several distinct advantages over fluorescentlabels. These advantages include vastly more sensitive detection,chemical uniformity, and the absolute resistance of the SERS activity tophotobleaching or photodegradation. A further benefit of using SACNs asoptical tags is the ease with which individual SACNs having differentSERS activities may be resolved from one another. At least twentydifferent SACNs are resolvable from one another using a simple Ramanspectroscope. This enables multiplexed assays to be performed using apanel of different SACNs, each having a unique and distinguishable SERSactivity. It also provides for, when SACNs are formed into groups, avery large number of unique SACN combinations with which objects can belabeled.

A surface-enhanced spectroscopy-active composite nanoparticle (SACN)contains a surface-enhanced spectroscopy (SES)-active (e.g.,SERS-active) metal nanoparticle that has attached to or associated withits surface one or more spectroscopy-active (e.g., Raman-active)molecules (alternately referred to herein as “analytes”). This complexof Raman-enhancing metal and analyte is then coated or encapsulated byan encapsulant. A SACN typically has a diameter of less than 200 nm or,alternatively, less than 100 nm. Each SACN is identified by the distinctRaman spectrum of its Raman-active analyte. Note that a metalnanoparticle is referred to as “surface-enhanced spectroscopy active”because it acts to enhance the spectroscopic signal of the associatedanalyte; it is the signal of the analyte, which is inherentlyspectroscopy active, that is being measured.

SACNs can be produced by growing or otherwise placing a shell of asuitable encapsulant over a SERS-active metal nanoparticle core withassociated Raman-active analyte. The metal nanoparticle core can be, forexample, a gold or silver sphere of between about 20 nm and about 200 nmin diameter. In another embodiment, the metal nanoparticle has adiameter of between about 40 nm and about 100 nm. In an alternativeembodiment, the metal nanoparticle is an oblate or prolate metalspheroid. For SERS using red incident light (˜633 nm), a suitable SERSresponse can be obtained with 63 nm diameter gold nanoparticles, butother particle diameters can also be employed. Typically, a SACNcontains only one metal nanoparticle, but more than one particle can beencapsulated together if desired. In a collection or plurality of SACNs,some may have one metal nanoparticle and some more than one metalnanoparticle. In this collection of particles, at least some of thedetected Raman signal originates from the particle or particlescontaining only one metal nanoparticle. Because the Raman signalintensity is a substantially linear function of the number ofRaman-active analytes, limiting a group of SACNs to a known number ofmetal nanoparticles and adsorbed analyte molecules allows estimation ofthe number of particles from the signal intensity of a group ofparticles.

The metal nanoparticles can contain any SES-active metal, i.e., anymetallic substance for which chemical enhancement, electromagneticenhancement, or both, is known in the art. For example, the metalnanoparticles can contain Au, Ag, or Cu. Other suitable metals include,but are not limited to, Na, K, Cr, Al, or Li. The metal particles canalso contain alloys of metals. In one embodiment, the metal nanoparticleconsists of a core (of pure metal or an alloy) overlaid with at leastone metal shell. In this case, the composition of the outer layer can bechosen to maximize the intensity of the Raman signal from the analyte.

Metal nanoparticles of the desired size can be grown as metal colloidsby a number of techniques well known in the art. For example, chemicalor photochemical reduction of metal ions in solution using any number ofreducing agents has been described. Likewise, nanoparticle syntheseshave been carried out in constrained volumes, e.g. inside a vesicle.Nanoparticles can also be made via electrical discharge in solution.Dozens of other methods have been described, dating back to themid-1800's.

The Raman-active analyte can be any molecular species having ameasurable SERS spectrum. Whether or not a spectrum is measurable maydepend upon the instrument with which the spectrum is acquired and theamount of adsorbed analyte. In general, however, a measurable spectrumis one that is detectable in the presence of the metal nanoparticle andencapsulant and can be recognized as characteristic of the particularanalyte. Typically, the maximum intensity of the SERS spectrum of theanalyte is substantially greater than that of the particle without theanalyte (i.e., the metal nanoparticle and encapsulant). Aromaticmolecules often have measurable Raman spectra. Examples of aromaticmolecules suitable for use as analytes in embodiments of SACNs include,but are not limited to, trans-1,2-bis(4-pyridyl)ethylene (BPE),pyridine, 2-mercaptopyridine, furonitrile, imidazole, andpara-nitroso-N,N′-dimethylaniline (p-NDMA).

Those skilled in the art will recognize that there is a great deal oflatitude in the composition of an analyte that yields a distinct Ramanspectrum. For example, in some embodiments, the analyte is not amolecule: it can be a positively or negatively charged ion (e.g., Na⁺ orCN⁻). If the analyte is a molecule, it can be neutral, positivelycharged, negatively charged, or amphoteric. The analyte can be a solid,liquid or gas. Non-molecular species such as metals, oxides, sulfides,etc. can serve as the Raman-active species. Any species or collection ofspecies that gives rise to a unique Raman spectrum, whether solid,liquid, gas, or a combination thereof, can serve as the analyte.Examples easily number in the many millions and include but are notlimited to Hg, dimethylformamide, HCl, H₂O, CN⁻, polypyrrole,hemoglobin, oligonucleotides, charcoal, carbon, sulfur, rust,polyacrylamide, citric acid, and diamond. In the case of diamond, theunique phonon mode of the particle can be used. For hemoglobin, only theporphyrin prosthetic group exhibits significant Raman activity; thus,complex substances can be used as the analyte if only part of themolecular or atomic complexity is present in the Raman spectrum.

The analyte can also be a polymer to which multiple Raman-activemoieties are attached. In this case, differentiable SACNs contain thesame polymer serving as the analyte, but the polymers have differentattached moieties yielding different Raman spectra. The polymer backbonedoes not itself contribute to the acquired Raman spectrum. In oneembodiment, the polymer is a linear chain containing amine groups towhich Raman-active entities are attached. Alternatively, the polymer canbe a dendrimer, a branched polymer with a tightly controlled tree-likestructure, with each branch terminating in a Raman-active species. Asuitable dendrimer structure has four generations of branchesterminating in approximately 45 Raman-active entities.

Note that SACNs that give rise to unique Raman spectra can be considereddifferent even if the analyte is essentially the same. For example, theRaman spectrum of a cationic polymer charge compensated by anions canchange depending on the choice of counter ion. A panel of differentiableSACNs can be formed using this polymer as a component of the analyte;each unique SACN has the polymer charge-compensated by a differentanion, thereby endowing each SACN with a unique Raman spectrum. Inaddition, a given analyte may have different Raman shifts on differentSERS-active layers, and differentiable SACNs can be formed using thesame analyte sandwiched between layers of different metals. For example,p-NDMA has different Raman shifts on gold and silver surfaces.Alternatively, one or more bands in the Raman spectrum of an analyte maybe dependent on the density of the analyte in the SACN. SACNs formedwith different densities of the same analyte are thereforedifferentiable from one another.

As will be apparent to one skilled in the art, characteristics of somesuitable Raman-active analytes are (i) strong Raman activity, whichminimizes the number of molecules needed to provide a given signalstrength; and (ii) simple Raman spectrum, which allows a large number ofunique SACNs to be distinguished when used simultaneously.

The Raman-active analyte can form a sub-monolayer, a complete monolayer,or a multilayer assembly on the surface of the metal nanoparticle core.A Raman-active analyte can be a single species of Raman-active molecule,a mixture of different species of Raman-active molecules, or a mixtureof Raman-active molecules and molecules without measurable Ramanactivity.

Typically, the encapsulant does not measurably alter the SERS activityof the metal nanoparticle or the Raman spectrum of the analyte. In analternative embodiment, however, the encapsulant can have a measurableeffect without adding significant complexity to the Raman spectrum. Theencapsulant can be readily modified in order to attach molecules,including biomolecules, to its exterior surface. Suitable encapsulantsinclude, but are not limited to, glass, polymers, metals, metal oxides(such as TiO₂ and SnO₂), and metal sulfides. The encapsulation iscarried out after, or during, adsorption to the core nanoparticle of theRaman-active analyte that is to provide the Raman activity of the SACN.In this way, the Raman-active analyte is sequestered from thesurrounding solvent. Such a configuration provides the metalnanoparticle core with stable SERS activity. Alternatively, it may notbe necessary to sequester the analyte completely, in which case theencapsulant does not completely surround the metal nanoparticle andanalyte.

The thickness of the encapsulant can be easily varied depending on thephysical properties required of the SACN. For example, coatings that aretoo thick—on the order of 1 micron or more—might preclude obtainingintense Raman spectra. Coatings too thin might lead to interference inthe Raman spectrum of the analyte by molecules on the encapsulantsurface. Raman scattering intensity decreases exponentially withdistance between analyte and nanoparticle surface; beyond 2 nm, theenhancing effect is negligible. An encapsulant that is at least thisthick prevents interference in the spectrum from molecules on theoutside of the SACN. Physical properties such as sedimentationcoefficient will clearly be affected by the thickness of encapsulant. Ingeneral, the thicker the encapsulant, the more effective thesequestration of the Raman-active analyte(s) on the metal nanoparticlecore from the surrounding solvent. One suitable thickness range of theencapsulant is between about 1 nm and about 40 nm. Alternatively, theencapsulant can be between about 5 nm and about 15 nm thick. Anothersuitable thickness range is between about 10 nm and about 20 nm.

In one embodiment of the invention, the encapsulant is glass (e.g.,SiO_(x)). To encapsulate in glass, the metal nanoparticle cores arepreferably treated first with a glass primer (that is, a material thatcan lead to growth of a uniform coating of glass, or can improveadhesion of the glass coat to the particle, or both). Glass is thengrown over the metal nanoparticle by standard techniques well known inthe art. The resulting SACNs are referred to as glass analyte-loadednanoparticles (GANs). For GANs, a suitable glass thickness ranges fromabout 1 nm to about 40 nm or, alternatively, between about 10 nm andabout 20 nm. In one embodiment, the GAN contains a 60 nm diameter goldparticle encapsulated by a 16 nm thick shell of glass. In an alternativeembodiment, the encapsulant is TiO₂, which is chemically similar to SiO₂and commonly used in many industries.

It may be desirable to separate true SACNs from free particles ofencapsulant that were not nucleated around a metal nanoparticle. Suchseparation improves the SERS activity of the nanoparticle preparationbecause free encapsulant particles are not SERS active. For example,GANs can be separated from free glass particles by size-exclusioncentrifugation in 50% glycerol.

Note that glass and many other materials contain functional groupsamenable to molecular attachment. For example, immersion of glass inbase allows covalent attachment of alkyl trichlorosilanes or alkyltrialkoxysilanes, with additional functionality available on the end ofthe alkyl group. Thus, glass surfaces can be modified with all forms ofbiomolecules and biomolecular superstructures including cells, as wellas oxides, metals, polymers, etc. Likewise, surfaces of glass can bemodified with well-organized monomolecular layers. In short, glasscoatings support essentially any and all forms of chemicalfunctionalization (derivatization). This is equally true for manydifferent forms of encapsulant, so that SACNs can be affixed to anyspecies with chemically reactive functionality. All chemical functionalgroups are reactive under certain conditions. There is thus nolimitation to the species that can be immobilized on the encapsulantsurface.

The optimization of the dimensions of the SACNs is readily accomplishedby one skilled in the art. Accordingly, one might alter the compositionof the particle, or its size and shape, in accordance with the inventionto optimize the intensity of the Raman signal. Indeed, it is known thatcore-shell nanoparticles (i.e. Au/AuS) support SERS and have verydifferent optical properties compared to pure metal nanoparticles.Likewise, it is known that SERS from prolate spheroids is enhancedrelative to spheres with the same major axis. It is further known thatsingle particle enhancements are strongly wavelength-dependent. Thus,one might “tune” the particle size, shape, and composition to achievemaximum signal for a given excitation wavelength.

One embodiment of the present invention contemplates the formation of apanel of at least 20 different SACNs, each having a unique SERSspectrum. This panel is referred to herein as a collection ofdistinguishable particles. Because the Raman bands of many molecules areextremely narrow (for example, CN⁻ is less than 1 nm at FWHM), it ispossible to synthesize a panel of SACNs, each containing a Raman analytethat is spaced 20 wavenumbers away in the spectrum from its closestneighbor. For example, a GAN with ¹³CN as the analyte is easilydistinguished from a GAN with ¹²CN as the analyte, and as well easilydistinguishable from one with C¹⁵N. In this way, it is possible to form540 distinct and easily resolvable peaks in a single Raman spectrum at633 nm from 300 to 3000 cm⁻¹ using a spectrograph to spread the photonsand a CCD camera as a detector. In general, Raman-active analytes can beused that have isotopic compositions distinct from naturally abundantspecies. For example, as described above, ¹³CN is completely resolvablefrom any natural ¹²CN that may be present in the background. Of course,those skilled in the art will recognize that combinations of isotopes aswell as ratios of isotopes can be equally effectively used to identifyunique SACNs.

Raman experiments with GANs or other SACNs can also be carried out withvisible or near-IR irradiation, make use of Raman bands from 100 cm⁻¹ to5000 cm⁻¹, employ any form of monochromator or spectrometer to spatiallyor temporally resolve photons, or employ any form of photon detector.This arrangement facilitates the synthesis of panels of at least 10resolvable SACNs, and provides ample bandwidth for literally hundreds ofpanels of SACNs.

Although the SERS activity of each population of SACNs in the panel isunique, the other properties of the SACNs are kept uniform across thepanel. Because the SERS activity of each SACN is sequestered from thesurrounding milieu by the encapsulant, individual populations do nothave different solvent or storage requirements. Also, each SACN has thesame exterior shell, simplifying the choice of chemistry either forattachment of molecules to the SACNs or attachment of the SACNs to solidsupports.

While the examples above have focused on Raman scattering, and inparticular surface-enhanced Raman scattering as the detection mechanism,a number of analogous methods can apply equally well and are includedwithin the scope of the present invention. For example, one can employ aresonantly-excited analyte, thus making the technique surface-enhancedresonance Raman scattering (SERRS). One could also take advantage ofexisting methods of surface-enhanced infrared absorption spectra (SEIRA)from nanoscale roughened surfaces. Likewise, surface-enhanced hyperRamanscattering (SEHRS) also occurs at nanoscale roughened metal surfaces,and this technique as well as its resonant analogue SEHRRS can beemployed. Note that for a given molecule, with either 3N-5 or 3N-6unique vibrations, where N is the number of atoms, all vibrations can befound in either the Raman, hyperRaman, or infrared spectrum. Indeed,identification of certain SACNs could rest on a combination of opticalinterrogation methods, including SERS, SERRS, SEIRA, SEHRS and SEHRRS.

Note also that a significant amount of (Rayleigh) light scattering isknown to occur from particles with dimensions at least 1/10 the excitingwavelength, thus creating the possibility that Rayleigh or hyperRayleighscattering could be used in identification of SACNs. Moreover,combinations of elastic scattering (e.g. Rayleigh), inelastic scattering(e.g. Raman), and absorption (e.g. IR) could be used to identifyparticles.

Use of SACNs

The SACNs provided by embodiments of the present invention can be usedin virtually any application in which a detectable tag or label isrequired. In some embodiments, SACNs are used in biological and chemicalassays as replacements for standard fluorescent tags. Indeed, SACNspossess a number of characteristics that make them far superior to priorart optical tags based on fluorophores. For example, assays usingfluorophore detection are commonly hampered by the presence ofautofluorescence and other background effects. In addition, many assaysrequire use of a number of different fluorophores; differentfluorophores commonly require different attachment chemistries and havedifferent environmental requirements and sensitivities. Particularlynoteworthy is the quenching of fluorescent activity that is observedwhen some fluorophores are conjugated to proteins. Finally, irreversiblephotodegradation resulting from the creation of a triplet or singletexcited state, followed by a non-reversible chemical reaction thatpermanently eliminates the excited state, places a severe limitation onthe sensitivity of detection. By contrast, SACNs cannot be photobleachedor photodegraded, they have uniform chemical and physical properties,and they can be readily resolved from the background. Perhaps mostimportantly, SACN detection is significantly more sensitive thanfluorophore detection. Indeed, it is possible to tag a single moleculewith a single SACN, and then detect the presence of that molecule usingRaman spectroscopy. Such simple single molecule resolution is withoutparallel in the fluorophore detection art.

An example of a biological assay in which SACNs can be used as opticaltags is the sandwich immunoassay. In sandwich assays, a target to bedetected is captured by a solid surface. An antibody (or other ligand)to the same target is attached to a SACN, and then contacted with thesolid support. The presence of the SACN SERS signal at the solid supportindicates the presence of the antigen. In general, SACNs can beconjugated to any molecule that is used to detect the presence of aspecific target in an assay.

In a specifically contemplated embodiment, SACNs are conjugated tonucleic acid molecules. In this way, they can be used in virtually anyassay known in the art that detects specific nucleic acid sequencesusing optically-tagged nucleic acid probes.

SACNs are especially suitable for multiplexed chemical assays in whichthe identity of SACNs encodes the identity of the target of the assay.Prior art multiplexed assays that use fluorophores to encode targetidentity are subject to a number of severe constraints imposed by thephysical and chemical properties of the fluorophores. Specifically,different fluorophores have different excitation maxima, so coincidentexcitation of multiple fluorescent tags is not possible. Moreover,fluorescence emission occurs in broad spectral bands, so the bands fromone fluorophore often overlap with those of another. As a result,resolving even three different fluorescence activities requiressophisticated optics to separate and then detect the individual emissionwavelengths. Because of these problems, multiplexed assays that usefluorophores rely on positional information to reveal target identity.Often, multiplexed assays with fluorophores use a solid support on whichligands are arranged in defined positions. The location of fluorophoresignal reveals the identity of the target; the size of the fluorophoresignal at that location indicates the amount of the target. However, thesynthesis of solid supports with reagents localized at specificpositions is expensive and time-consuming. Also, there are limits on thenumber of features that may be defined on a single surface.

By contrast, the SACNs of the present invention offer remarkablespectral diversity and resolvability. As a result, SACNs can be used inmultiplexed assays to yield quantitative and qualitative informationwithout requiring the position-specific localization of reagents. EachSACN coupled to a target-specific reagent can encode the identity ofthat specific target, and the intensity of a particular Raman signalreveals the quantity of that target. For example, in the sandwichimmunoassays described above, the identity of targets captured on thesolid support can be determined by using a different flavor of SACN foreach target.

Although SACNs are perfectly suited for use in multiplexingapplications, they need not be used to encode identity in this manner.They can be used simply as replacements for fluorophores in multiplexedassays in which reagents are localized to specific positions on solidsupports. When used in this way, the SACNs offer vastly more sensitivetarget detection than fluorophores.

In other embodiments, SACNs serve as tags for labeling objects ormaterials, e.g., for anti-counterfeiting or authentication purposes, orfor encoding the history of an object moving through a manufacturingprocess or supply chain. In these applications, one or more SACNs areassociated with an object or material and later “read” by Ramanspectroscopy to determine the identity of the particle or particles andobtain information about the tagged object. The acquired spectrum can becompared to a reference spectrum or to a spectrum of the particlesacquired before they were associated with the object. If necessary,suitable corrections can be made to account for background emission fromthe object. Authentication can occur at any desired point during thelifetime of the object, e.g., upon receipt of a manufactured object by aretailer or upon sale of an antique object.

Each SACN or group of SACNs, with its unique Raman spectrum, correspondsto or represents a particular piece of information. Any type ofinformation can be represented by a SACN, depending upon theapplication. For example, a SACN or group of SACNs can represent anindividual object such as an item of sports memorabilia, a work of art,an automobile, or the item's owner or manufacturer; a class of objects,such as a particular formulation of pharmaceutical product; or a step ofa manufacturing process. The information represented by a particularRaman spectrum or SACN type can be stored in a database, computer file,paper record, or other desired format.

The small, robust, non-toxic, and easily-attachable nature of SACNsallows their use for tagging virtually any desired object. The trackedobject can be made of solid, liquid, or gas phase material or anycombination of phases. The material can be a discrete solid object, suchas a container, pill, or piece of jewelry, or a continuous or granularmaterial, such as paint, ink, fuel, or extended piece of, e.g., textile,paper, or plastic, in which case the particles are typically distributedthroughout the material.

Examples of specific materials or objects that can be tagged with SACNsinclude, but are not limited to:

-   -   Packaging, including adhesives, paper, plastics, labels, and        seals    -   Agrochemicals, seeds, and crops    -   Artwork    -   Computer chips    -   Cosmetics and perfumes    -   Compact disks (CDs), digital video disks (DVDs), and videotapes    -   Documents, money, and other paper products (e.g., labels,        passports, stock certificates)    -   Inks, paints, and dyes    -   Electronic devices    -   Explosives    -   Food and beverages, tobacco    -   Textiles, clothing, footwear, designer products, and apparel        labels    -   Polymers    -   Hazardous waste    -   Movie props and memorabilia, sports memorabilia and apparel    -   Manufacturing parts    -   Petroleum, fuel, lubricants, oil    -   Pharmaceuticals and vaccines

Particles can be associated with the material in any way that maintainstheir association at least until the particles are read. Depending uponthe material to be tagged, the particles can be incorporated duringproduction or associated with a finished product. Because they are sosmall, the particles are unlikely to have a detrimental effect on eitherthe manufacturing process or the finished product. The particles can beassociated with or attached to the material via any chemical or physicalmeans. For example, particles can be mixed with and distributedthroughout a liquid-based substance such as paint, oil, or ink and thenapplied to a surface. They can be wound within fibers of a textile,paper, or other fibrous or woven product, or trapped between layers of amulti-layer label. The particles can be incorporated during productionof a polymeric or slurried material and bound during polymerization ordrying of the material. Additionally, the surfaces of the particles canbe chemically derivatized with functional groups of any desiredcharacteristic, as described above, for covalent or non-covalentattachment to the material. When the particles are applied to a finishedproduct, they can be applied manually by, e.g., a pipette, orautomatically by a pipette, spray nozzle, or the like. Particles can beapplied in solution in a suitable solvent (e.g., ethanol), which thenevaporates.

SACNs have a number of inherent properties that are advantageous fortagging and tracking applications. They offer a very large number ofpossible codes. For example, if a panel of SACNs is constructed with 20distinguishable Raman spectra, and an object is labeled with two SACNs,there are 20*19/2=190 different codes. If the number of particles perobject is increased to 5, there are 15,504 possible codes. Ten particlesper object yields 1.1×10⁶ different codes. A more sophisticatedmonochromator increases the number of distinguishable Raman spectra to,e.g., 50, greatly increasing the number of possible codes.

SACNs can be identified using a conventional Raman spectrometer. Infact, one benefit of using SACNs is the versatility of excitationsources and detection instrumentation that can be employed for Ramanspectroscopy. Visible or near-IR lasers of varying sizes andconfigurations can be used to generate Raman spectra. Portable,handheld, and briefcase-sized instruments are commonplace. At the sametime, more sophisticated monochromators with greater spectral resolvingpower allow an increase in the number of unique taggants that can beemployed within a given spectral region. For example, the capability todistinguish between two Raman peaks whose maxima differ by only 3 cm⁻¹is routine.

Typically, if a suitable waveguide (e.g., optical fiber) is provided fortransmitting light to and from the object, the excitation source anddetector can be physically remote from the object being verified. Thisallows SACNs to be used in locations in which it is difficult to placeconventional light sources or detectors. The nature of Raman scatteringand laser-based monochromatic excitation is such that it is notnecessary to place the excitation source in close proximity to theRaman-active species.

Another characteristic of SACNs is that the measurement of their Ramanspectra need to strictly be confined to “line of sight” detection, aswith, e.g., fluorescent tags. Thus their spectrum can be acquiredwithout removing the particles from the tagged object, provided that thematerial is partially transparent to both the excitation wavelength andthe Raman photon. For example, water has negligible Raman activity anddoes not absorb visible radiation, allowing SACNs in water to bedetected. SACNs can also be detected when embedded in, e.g., clearplastic, paper, or certain inks.

SACNs also allow for quantitative verification, because the Raman signalintensity is an approximately linear function of the number of analytemolecules. For standardized particles (uniform analyte distribution),the measured signal intensity reflects the number or density ofparticles. If the particles are added at a known concentration, themeasured signal intensity can be used to detect undesired dilution ofliquid or granular materials.

SACNs are chemically and biologically inert, and a glass coating givesthe particles charge, flow, and other physical properties similar tothose of SiO₂ particles commonly used as excipients in pills, vitamins,and a wide variety of other materials. A TiO₂ coating on SACNs allowthem to be used in the very large number of materials that currentlycontain TiO₂, such as papers, paints, textiles, and apparel.

Because of their submicron size, SACNs can be added to fluids withoutchanging the fluid properties significantly or affecting the fluidhandling equipment. For example, the particles can flow through narrowtubes and be expelled out nozzles without clogging lines or orifices.

SACNs are also non-toxic and can be ingested safely by humans and otheranimals. This enables their tagging of pharmaceutical products, foodproducts, and beverages (e.g., wine). Particles are comparable in sizeto the excipients normally used as vehicles for drugs.

The following examples are offered by way of illustration and not by wayof limitation.

WORKING EXAMPLES Working Example 1

Synthesis of Glass-Coated Analyte-Loaded Nanoparticles (GANs)

Materials: Water used for all preparations was 18.2 MΩ, distilledthrough a Barnstead nanopure system. Snake skin dialysis tubing, 3,500MWCO, was purchased from Pierce. 3-aminopropyltrimethoxysilane (APTMS),3-mercaptotrimethoxysilane (MPTMS), and3-mercaptopropylmethyldimethoxysilane (MPMDMS) were obtained from UnitedChemical. HAuCl₄.3H₂O, trisodium citrate dihydrate, sodium hydroxide,trans-1,2-bis(4-pyridyl)ethylene (BPE), pyridine, 2-mercaptopyridine,sodium silicate, tetraethyl orthosilicate (TEOS), and ammonia wereobtained from Sigma-Aldrich. BPE was recrystallized several times beforeuse. Dowex cation exchange resin (16-40 mesh) was obtained from J. T.Baker. Pure ethyl alcohol (EtOH) was purchased from Pharmco.

Colloid preparation: 12-nm colloidal Au (nearly spherical, with astandard deviation less than 2 nm) was prepared from HAuCl₄.3H₂O reducedby citrate as described in Grabar et al, Analytical Chemistry 67:735-743(1995), incorporated herein by reference in its entirety.

Colloid >12 nm was prepared as follows: 3 ml of 12 mM HAuCl₄ was addedfor every 97 ml of H₂O. The solution was then brought to a boil undervigorous stirring and 1 ml of 12-nm Au colloid as a seed and 0.5 ml of1% sodium citrate per 100 ml of HAuCl₄ solution was added and boiled for10 minutes. The size of the resulting particles was determined bytransmission electron microscopy using Gatan or NIH Image software.Finally, the citrate ions surrounding the Au colloid were removed withdialysis, 7 exchanges of at least 4 hours each.

GANs preparation: All reactions were performed in plastic Erlenmeyerflasks. Any amount of colloid could be used in a preparation and thesubsequent reactants added in appropriate amounts based on the surfacearea and concentration of the Au colloid.

A typical experiment used 25 ml of dialyzed, 50-nm, 0.17 nM Au colloid.The pH of the colloid was adjusted from 5 to 7 with the addition of 50μL of 0.1 M NaOH. The colloid was rendered vitreophilic with theaddition 125 μL of 0.5 mM MPTMS (or APTMS, or MPMDMS). After 15 minutesof magnetic stirring, 167 μL of a 0.5 mM solution of the Raman tag (BPE,pyridine, or 2-mercaptopyridine) was added. During another 15 minuteperiod of stirring, a 0.54% solution of active silica was prepared bymixing 1 g of sodium silicate with 50 ml of 3 M NaOH and lowering the pHto 10 with cation exchange resin. One ml of the active silica was addedand the resulting solution was approximately pH 9. The solution remainedstirring for 15 minutes and then was allowed to stand.

After a 24 hour period, 100 ml of EtOH was added to the solution toproceed with silica growth via the method described in Stöber et al, J.Colloid Interface Sci. 26: 62 (1968), incorporated herein by referencein its entirety. Growth of ˜4 nm of additional glass shell wasaccomplished with the addition of 15 μL of TEOS and 125 μL of ammonia.The reaction was stirred for 15 minutes and then allowed to stand for atleast 12 hours. The addition of TEOS and ammonia was continued until thedesired shell thickness was obtained.

Working Example 2

Transmission Electron Microscopy of GANs

Transmission electron microscopy (TEM) images were taken of preparationsof GANs; these TEM images illustrate the uniformity of GANspreparations. FIG. 1A shows GANs containing 35 nm Au cores with 40 nmglass. FIG. 1B shows 60 nm Au cores with 16 nm glass. FIG. 2 illustrates35 nm Au, 8 nm glass GANs following centrifugation through a 50%glycerol solution.

Working Example 3

Demonstration of the Sequestration of the Metal Core from Solvent

For GANs to function in diverse chemical environments, it is necessarythat the Raman-active analyte be sequestered from the surroundingsolvent. To demonstrate this sequestration, one may look at diffusionrates through the glass network. This is done by monitoring the rate atwhich aqua regia (3 HCl: 1 HNO₃) is able to etch out the Au core of aGAN. FIG. 3 demonstrates one such experiment for a batch of GANsparticles with a 35 nm Au core, and 8 nm shell of glass. To 500 μl of≈0.17 nM GANs was added 200 μl of an etch solution (50 μl HNO₃ and 150μl HCl). The absorbance of the solution was measured (λ_(max) 546 nm) atvarious times after addition of the etch solution. Etching of the goldcore results in a decrease in the absorbance; this is plotted in FIG. 3A(the time after the addition of the etch solution is indicated). Therate of Au etching is shown in FIG. 3B as a plot of absorbance versustime in etch solution (right). Additional studies performed by theinventors have shown that etching of a Au core by aqua regia does notoccur with a 20 nm glass shell over a four hour time period.

Working Example 4

SERS Spectra of GANs Particles

GANs containing a 40 mm Au core coated withtrans-1,2-bis(4-pyridyl)ethylene (BPE) encapsulated in 4 nm of glasswere synthesized and examined by Raman spectroscopy. The Raman spectrumobtained using 20 mW of 632.8 nm excitation, with a 3 mm lens and 30second integration is plotted in FIG. 4. Trace A on the graph shows thecharacteristic BPE Raman signal; trace B shows the Raman signal from thesame particles without the BPE analyte. It can be seen that the GANswithout the BPE analyte give essentially no Raman signal.

Working Example 5

Confinement of the Raman-Active Analyte to the Metal Core of GANs byGlass Encapsulation

FIG. 5 shows the Raman spectrum of a suspension of GANs comprising 40 nmAu coated with trans-1,2-bis(4-pyridyl)ethylene (BPE)/4 nm glass (TraceA); supernatant from a first centrifugation of the GANs (Trace B); andsupernatant from a second centrifugation of the GANs (Trace C). It canbe seen that the BPE signal does not leave the GAN during eachcentrifugation step, indicating that the BPE has adhered to the Au coreand is tightly sequestered there by glass encapsulation.

Working Example 6

Comparison of SERS Spectra of Raman-Active Analytes on GANs with OtherSERS Substrates

GANs (80 nm Au core/20 nm glass) containing 2-mercaptopyridine as theRaman-active analyte were analyzed by Raman spectroscopy using 25 mW of632.8 nm excitation with a 3 mm lens and 60 seconds of integration. TheRaman spectrum of the GANs preparation was then compared with the Ramanspectrum obtained when a 50 mM solution of 2-mercaptopyridine isabsorbed onto a conventional three-layer SERS substrate (25 mW 632.8 nmexcitation, 3 mm lens, 30-seconds integration). FIG. 6 shows the twoRaman spectra. It can be seen that the two spectra have identicalfeatures and intensities, illustrating that the metal nanoparticles ofthe GANs are effective SERS substrates.

Working Example 7

SERS Spectra of GANs with Mixtures of Raman-Active Analytes

SERS spectra of the following four flavors of GANs particles wereobtained using 26 mW of 632.8 nm excitation, a 3-mm lens, and 30-secondintegration: (A) GANs tagged with furonitrile; (B) GANs tagged withfuronitrile (66%) and cyanoacetic acid (33%); (B) GANs tagged withfuronitrile (33%) and cyanoacetic acid (66%); and (D) GANs tagged withcyanoacetic acid. The percentages indicated are the relativeconcentrations of each compound in the tagging solution added. FIG. 7shows that the furonitrile and cyanoacteic acid have relatively the samesignal intensity and have similar spectral profiles. The fact that thespectra of B and C are very similar to the spectrum of D indicates thatcyanoacetic acid has a better affinity for the Au nanoparticle thanfuronitrile.

Working Example 8

SERS Spectra of GANs Tagged with Imidazole (IM) andtrans-4,4′-bis(pyridyl)ethylene (BPE)

GANs (40 nm Au core/4 nm glass) were tagged with either (a)trans-1,2-bis(4-pyridyl)ethylene (BPE-GANs) or (b) imidazole (IM-GANs).SERS spectra of these Raman-active analytes are shown in FIG. 8, alongwith the SERS spectrum of untagged GANs (c) of the same dimensions.BPE-GANs and IM-GANs both show the characteristic Raman bands of theirrespective Raman-active analytes; untagged GANs do not show these bands.

Working Example 9

Tagging Paper with GANs

GANs were prepared from 12 nm-diameter gold particles coated withtrans-1,2-bis(4-pyridyl)ethylene (BPE) orpara-nitroso-N,N′-dimethylaniline (p-NDMA) as described in WorkingExample 1. Encapsulation was completed with a single addition of TEOSand ammonia. Particles were stored in ethanol as prepared (at aconcentration of approximately 1 nM).

The particle solutions were mixed and three spots each of approximately10-20 μl of the resulting solution were pipetted onto yellow and whitesheets of conventional paper. After evaporation of ethanol, spots werevisible on both sides of the paper, indicating that the particles hadpenetrated the sheets.

Raman spectra of the spots were acquired using approximately 20 mW of633 nm excitation with a 3 mm lens and 30 seconds of integration. FIG.9A shows a representative spectrum on white paper and FIG. 9B on yellowpaper. Both the yellow and white paper displayed high levels ofbackground fluorescence. However, characteristic peaks of BPE at about1200, 1610, and 1640 cm⁻¹ were detectable over the background signal inboth cases.

It should be noted that the foregoing description is only illustrativeof the invention. Various alternatives and modifications can be devisedby those skilled in the art without departing from the invention.Accordingly, the present invention is intended to embrace all suchalternatives, modifications and variances which fall within the scope ofthe disclosed invention.

1. A method of tagging a material, comprising associating with saidmaterial a particle comprising: a surface-enhanced spectroscopy(SES)-active metal nanoparticle, a spectroscopy-active analyte, and anencapsulant surrounding said SES-active metal nanoparticle and saidspectroscopy-active analyte.
 2. The method of claim 1, wherein saidmetal nanoparticle comprises a metal selected from the group consistingof Au, Ag, Cu, Na, Al, and Cr.
 3. The method of claim 2, wherein saidmetal nanoparticle comprises Au.
 4. The method of claim 2, wherein saidmetal nanoparticle comprises Ag.
 5. The method of claim 1, wherein saidmetal nanoparticle has a diameter less than about 200 nm.
 6. The methodof claim 5, wherein said metal nanoparticle has a diameter between about20 nm and about 200 nm.
 7. The method of claim 6, wherein said metalnanoparticle has a diameter between about 40 nm and about 100 nm.
 8. Themethod of claim 1, wherein said encapsulant has a thickness less thanabout 1 micron.
 9. The method of claim 8, wherein said encapsulant has athickness between about 1 nm and about 40 nm.
 10. The method of claim 9,wherein said encapsulant has a thickness between about 5 nm and about 15nm.
 11. The method of claim 1, wherein said metal nanoparticle comprisesan alloy of metals selected from the group consisting of Au, Ag, Cu, Na,Al, and Cr.
 12. The method of claim 1, wherein said spectroscopy-activeanalyte forms a submonolayer coating on said metal nanoparticle.
 13. Themethod of claim 1, wherein said spectroscopy-active analyte forms amonolayer coating on said metal nanoparticle.
 14. The method of claim 1,wherein said spectroscopy-active analyte forms a multilayer coating onsaid metal nanoparticle.
 15. The method of claim 1, wherein saidencapsulant comprises a material selected from the group consisting ofglass, polymers, metals, metal oxides, and metal sulfides.
 16. Themethod of claim 1, wherein said encapsulant comprises a plurality ofmaterials selected from the group consisting of glass, polymers, metals,metal oxides, and metal sulfides.
 17. The method of claim 1, whereinsaid encapsulant comprises glass oxide (SiO_(x)).
 18. The method ofclaim 1, wherein said encapsulant comprises SiO_(x).
 19. The method ofclaim 1, wherein said surface-enhanced spectrum is obtained by a methodselected from the group consisting of SERS, SERRS, SEHRRS, and SEIRA.20. The method of claim 1, wherein said spectroscopy-active analyte isan aromatic analyte.
 21. The method of claim 1, wherein said materialcomprises an object.
 22. The method of claim 1, wherein said materialcomprises a liquid.
 23. The method of claim 22, wherein said material isselected from the group comprising ink, paint, and oil.
 24. The methodof claim 1, wherein said material comprises a material selected from thegroup comprising paper, textiles, polymers, and pharmaceuticals.
 25. Themethod of claim 24, wherein said material comprises paper.
 26. Themethod of claim 24, wherein said material comprises a textile.
 27. Themethod of claim 24, wherein said material comprises a polymer.
 28. Themethod of claim 24, wherein said material comprises a pharmaceutical.29. The method of claim 1 further comprising acquiring a SES spectrumfrom said material.
 30. A method of tagging a material, comprisingassociating with said material a plurality of particles, each of saidparticle comprising: a surface-enhanced spectroscopy (SES)-active metalnanoparticle, a spectroscopy-active analyte, and an encapsulantsurrounding said SES-active metal nanoparticle and saidspectroscopy-active analyte, wherein each of said particles comprises adistinct Raman spectrum.
 31. The method of claim 30, wherein saidparticle encode information relating to said tagged material.
 32. Themethod of claim 31, wherein said information relates to the manufactureof said tagged material.
 33. The method of claim 31, wherein saidinformation relates to the authenticity of said material.
 34. The methodof claim 31, wherein said information is stored in a location selectedfrom the group consisting of a database, a computer file, and a paperrecord.
 35. The method of claim 30 further comprising acquiring a SESspectrum from said material.